A highly sensitive LC-MS/MS method for quantitative determination of 7 vitamin D metabolites in mouse brain tissue

Abstract

Despite its critical role in neurodevelopment and brain function, vitamin-D (vit-D) homeostasis, metabolism and kinetics within the central nervous system remain largely undetermined. Thus, it is of critical importance to establish an accurate, highly sensitive and reproducible method to quantitate vit-D in brain tissue. Here, we present a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method and for the first time, demonstrate detection of seven major vit-D metabolites in brain tissues of C57BL/6J wild-type mice, namely: 1,25(OH)2D3, 3-epi-1,25(OH)2D3, 1,25(OH)2D2, 25(OH)D3, 25(OH)D2, 24,25(OH)2D3, and 24,25(OH)2D2. Chromatographic separation was achieved on a pentaflurophenyol column 3 mM ammonium formate with water/methanol [A] and methanol/isopropanol [B] phases. Detection was by positive-ion electrospray tandem mass spectrometry. We used calibration standards of each metabolite prepared in brain matrices to validate the detection range, precision, accuracy and recovery. Isotopically labelled analogues, 1,25(OH)2D3-d3, 25(OH)D3-C5 and 24,25(OH)2D3-d6, served as the internal standards for the closest molecular related metabolite in all measurements. The calibration range was between 1 fg/mL to 10 ng/mL with an LLOD and LLOQ of 10 fg/mL and 3 fg/mL, respectively. The intra-/inter-day precision and accuracy for measuring brain vit-D metabolites ranged between 0.12-11.53% and 0.28-9.11%, respectively. Recovery ranged between 99.06% and 106.9% for all metabolites. Collectively, the sensitivity and efficiency of our method supersedes previously reported protocols used to measure vit-D and to our knowledge, the first protocol to reveal the abundance of 25(OH)D2, 1,25(OH)D2 and 24,25(OH)2D2, in brain tissue of any species. This technique may be important in supporting the future advancement of pre-clinical research into the function of vit-D in neurophysiological, neuropsychiatric, and neurodegeneration.

Vitamin D metabolites and analytical challenges

“…The method exhibited excellent sensitivity with a LOQ and LOD of 10 fg mL −1 and 3 fg mL −1 , respectively. 122 Assessment of calcitriol is considerably more difficult due to its very low concentration in the body (pg mL −1 ) and the presence of significant amounts of other VD metabolites, which may interfere with the calcitriol measurements. In a recent study, Okai et al investigated two techniques (CLIA and LC-MS/MS) for determining 1,25(OH) 2 D in samples with 25(OH)D concentrations of more than 150 ng mL −1 .…”